Cryptococcus neoformans is an encapsulated pathogenic yeast that causes disease in humans and other animals. Currently, the majority of human patients with cryptococcosis have diseases or treatments that cause suppression of cell-mediated immunity. During the last 20 years, there has been a dramatic increase in the incidence of cryptococcosis that mirrors the increase in HIV infections. AIDS patients are exquisitely vulnerable to opportunistic fungal infections. It has been estimated that 7-10% of patients with AIDS will contract cryptococcosis, and if left untreated, the disease is fatal. It is a leading cause of death in immunocompromised patients. Caspofungin, a new echinocandin (1,3)beta-glucan synthase inhibitor, is effective against many species of fungi and is well tolerated, safe, and effective; in contrast to the azoles, it is fungicidal and would be extremely useful in the treatment of primary cryptococcosis. However, its use to treat C. neoformans infections is precluded since the organism is resistant to caspofungin both in vitro and in vivo. This result is puzzling given that the gene encoding (1,3)beta-glucan synthase is essential for C. neoformans growth. The reason(s) for resistance has not been determined, but is likely to involve one of the following: 1. The target itself ([1,3]beta-glucan synthase) is resistant to caspofungin; 2. Caspofungin is transported out of the cell by, for example, ABC transporters; or 3. Caspofungin is degraded either extra- and/or intra-cellularly. In this R21 proposal, we will determine the mechanism of resistance of C. neoformans to echinocandins with the goal of developing a series of hypotheses that can be tested in an R01. The eventual goal of that work will be to develop a strategy to circumvent resistance, for example, the design of a caspofungin derivative that is effective against C. neoformans. We propose three specific aims: One: Determine the in vitro resistance of C. neoformans (1,3)beta-glucan synthase activity to caspofungin. Two: Determine the involvement of ABC transporters in caspofungin resistance. Three: Determine whether caspofungin is degraded by C. neoformans. At the conclusion of these preliminary studies, we anticipate that we will have determined which of the three resistance mechanisms is the most important for C. neoformans resistance to glucan synthase inhibitors.